SARS-CoV-2 transmitters have more than three times higher viral loads than non-transmitters - Practical use of viral load for disease control.

BACKGROUND: Quantitative results of SARS-CoV-2 testing reported as viral load copies/mL can provide valuable information, but are rarely used in practice. We analyze whether viral load in the upper respiratory tract is correlated with transmission and disease course and how this information can be used in practice. STUDY DESIGN: Municipal Health Service (MHS) and clinical patients ≥18 years tested positive for SARS-CoV-2 with RT-PCR between June 1 and September 25, 2020 were included. Transmission was defined as an index having at least one contact tested positive. Test delay was defined as the time between symptom onset and SARS-CoV-2 testing. RESULTS: 683 patients were included (656 MHS and 27 clinical patients). The viral load was considerably lower among clinical patients compared to MHS patients: median log10 copies/mL 2.51 (IQR -1.52 - 6.46) vs 4.92 (IQR -0.54 - 8.26), p < 0.0001. However, the test delay was higher for clinical patients (median 7 [IQR 2 - 19] vs 3 [IQR 0 - 26] days, p < 0.0001). SARS-CoV-2 transmitters showed much higher viral loads than non-transmitters (log10 copies/mL 5.23 [IQR -0.52 - 8.26] vs 4.65 [IQR -0.72 - 8.00], p < 0.0001), but not for those with a test delay > 7 days. Higher viral loads were significantly correlated with older age and with more (severe) COVID-19 related symptoms. CONCLUSION: Indexes that transmitted SARS-CoV-2 had more than three times higher viral loads than non-transmitters. Viral load information can be useful during source and contact tracing to prioritize indexes with highest risk of transmission, taking into account the test delay.

Performance of the Diasorin SARS-CoV-2 antigen detection assay on the LIAISON XL.

BACKGROUND: The current reference standard to diagnose a SARS-CoV-2 infection is real-time reverse transcriptase polymerase chain reaction (RT-PCR). This test poses substantial challenges for large-scale community testing, especially with respect to the long turnaround times. SARS-CoV-2 antigen tests are an alternative, but typically use a lateral flow assay format rendering them less suitable for analysis of large numbers of samples. METHODS: We conducted an evaluation of the Diasorin SARS-CoV-2 antigen detection assay (DAA) compared to real-time RT-PCR (Abbott). The study was performed on 248 (74 qRT-PCR positive, 174 qRT-PCR negative) clinical combined oro-nasopharyngeal samples of individuals with COVID-19-like symptoms obtained at a Municipal Health Service test centre. In addition, we evaluated the analytical performance of DAA with a 10-fold dilution series of SARS-CoV-2 containing culture supernatant and compared it with the lateral flow assay SARS-CoV-2 Roche/SD Biosensor Rapid Antigen test (RRA). RESULTS: The DAA had an overall specificity of 100% (95%CI 97.9%-100%) and sensitivity of 73% (95%CI 61.3%-82.7%) for the clinical samples. Sensitivity was 86% (CI95% 74.6%-93.3%) for samples with Ct-value below 30. Both the DAA and RRA detected SARS-CoV-2 up to a dilution containing 5.2 × 102 fifty-percent-tissue-culture-infective-dose (TCID50)/ml. DISCUSSION: The DAA performed adequately for clinical samples with a Ct-value below 30. Test performance may be further optimised by lowering the relative light unit (RLU) threshold for positivity assuming the in this study used pre-analytical protocol . The test has potential for use as a diagnostic assay for symptomatic community-dwelling individuals early after disease onset in the context of disease control.

Successful containment of two vancomycin-resistant Enterococcus faecium (VRE) outbreaks in a Dutch teaching hospital using environmental sampling and whole-genome sequencing.

BACKGROUND: Vancomycin-resistant enterococci (VRE) may cause nosocomial outbreaks. This article describes all VRE carriers that were identified in 2018 at Elisabeth-Tweesteden Hospital, Tilburg, The Netherlands. AIM: To investigate the genetic relatedness of VRE isolates and the possibility of a common environmental reservoir using environmental sampling and whole-genome sequencing (WGS). METHODS: Infection control measures consisted of contact isolation, contact surveys, point prevalence screening, environmental sampling, cleaning and disinfection. VRE isolates were sequenced using a MiSeq sequencer (Illumina, San Diego, CA, USA), and assembled using SPAdes v.3.10.1. A minimal spanning tree and a neighbour joining tree based on allelic diversity of core-genome multi-locus sequence typing and accessory genes were created using Ridom SeqSphere+ software (Ridom GmbH, Münster, Germany). FINDINGS: Over a 1-year period, 19 VRE carriers were identified; of these, 17 were part of two outbreaks. Before environmental cleaning and disinfection, 55 (14%) environmental samples were VRE-positive. Fifty-one isolates (23 patient samples and 28 environmental samples) were available for WGS analysis. Forty-four isolates were assigned to ST117-vanB, five were assigned to ST17-vanB, and two were assigned to ST80-vanB. Isolates from Outbreak 1 (N=22) and Outbreak 2 (N=22) belonged to ST117-vanB; however, WGS showed a different cluster type with 257 allelic differences. CONCLUSION: WGS of two outbreak strains provided discriminatory information regarding genetic relatedness, and rejected the hypothesis of a common environmental reservoir. A high degree of environmental contamination was associated with higher VRE transmission. Quantification of environmental contamination may reflect the potential for VRE transmission and could therefore support the infection control measures.

Using eHealth to Support COVID-19 Education, Self-Assessment, and Symptom Monitoring in the Netherlands: Observational Study.

BACKGROUND: The coronavirus disease (COVID-19) situation demands a lot from citizens, health care providers, and governmental institutions. Citizens need to cope with guidelines on social interaction, work, home isolation, and symptom recognition. Additionally, health care providers and policy makers have to cope with unprecedented and unpredictable pressure on the health care system they need to manage. By providing citizens with an app, they always have access to the latest information and can assess their own health. This data could be used to support policy makers and health care providers to get valuable insights in the regional distribution of infection load and health care consumption. OBJECTIVE: The aim of this observational study is to assess people's use of an app to support them with COVID-19 education, self-assessment, and monitoring of their own health for a 7-day period. In addition, we aim to assess the usability of this data for health care providers and policy makers by applying it to an interactive map and combining it with hospital data. The secondary outcomes of the study were user's satisfaction with the information provided in the app, perceived usefulness of the app, health care providers they contacted, and the follow-up actions from this contact. METHODS: This observational cohort study was carried out at the nonacademic teaching hospital "Elisabeth Twee Steden" (ETZ) in Tilburg, Netherlands. From April 1, 2020, onwards ETZ offered the COVID-19 education, self-assessment, and symptom tracking diary to their already existing app for patient education and monitoring. RESULTS: Between April 1 and April 20, 2020, a total of 6194 people downloaded the app. The self-assessment functionality was used abundantly to check one's health status. In total, 5104 people responded to the question about severe symptoms, from which 242 indicated to suffer from severe symptoms. A total of 4929 people responded to the question about mild symptoms, from which 3248 indicated to suffer from these. The data was successfully applied to an interactive map, displaying user demographics and health status. Furthermore, the data was linked to clinical data. App users were satisfied with the information in the app and appreciated the symptom diary functionality. In total, 102 users reached out to a health care provider, leading to 91 contacts. CONCLUSIONS: Our study demonstrated the successful implementation and use of an app with COVID-19 education, self-assessment, and a 7-day symptom diary. Data collected with the app were successfully applied to an interactive map. In addition, we were able to link the data to COVID-19 screening results from the hospital's microbiology laboratory. This data could be used to support policy makers and health care providers to get valuable insights in the regional distribution of infection load and health care consumption. TRIAL REGISTRATION: Netherlands Trial Register NL8501; https://www.trialregister.nl/trial/8501.

Added diagnostic value of broad-range 16S PCR on periprosthetic tissue and clinical specimens from other normally sterile body sites.

AIMS: Evaluation of 16S PCR in addition to the standard culture to improve the pathogen detection rate in clinical specimens. METHODS AND RESULTS: Microbiological culture and direct 16S PCR was performed on specimens from suspected prosthetic joint infection patients (cohort-1) and on tissues and fluids from other normally sterile body sites (cohort-2). Based on clinical and microbiological data, the detection rate for both methods was assessed, assuming no superiority of either 16S PCR or culture. In cohort-1, 469 specimens were obtained. Culture was positive in 170 (36·2%) specimens, 16S PCR detected 70 (41·2%) of those pathogens. Additionally, 16S PCR detected pathogens in 13 of 299 (4·3%) culture-negative specimens. In cohort-2, pathogens were cultured in 52 of 430 (12·1%) specimens and 16S PCR revealed those pathogens in 32 (61·5%) specimens. 16S PCR detected pathogens in 31 of 378 (8·2%) culture-negative specimens. CONCLUSIONS: Overall, the yield with 16S PCR was low. For cohort-1 16S PCR detected pathogens in 4·3% of culture-negative specimens, where this was 8·2% for cohort-2. SIGNIFICANCE AND IMPACT OF THE STUDY: Although direct 16S PCR cannot replace culture, it may offer a valuable additional diagnostic option for detection of difficult to culture micro-organisms in culture-negative clinical specimens.

The clinical utility of JC virus antibody index measurements in the context of progressive multifocal leukoencephalopathy.

In natalizumab-treated patients without previous immunosuppressive treatment, the JCV antibody index is used to stratify PML risk. A high index value indicates that the risk to develop PML is significantly elevated, although probably about 99% of patients with this index value will not develop PML. This minireview aimed to provide an overview of the basic virology and immunology relevant to understanding JCV infections in MS patients, with a focus on what is presently known about antibodies to JCV and how they could be of use to predict and diagnose PML.

High cumulative JC virus seroconversion rate during long-term use of natalizumab.

BACKGROUND AND PURPOSE: John Cunningham virus (JCV) seropositivity is a risk factor for the development of natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS) patients. When JCV seronegative patients seroconvert, their risk of developing PML increases. Limited longitudinal data exist about the seroconversion rate amongst natalizumab-treated relapsing-remitting MS (RRMS) patients. Our objective was to evaluate the seroconversion rate in a large Dutch cohort of natalizumab-treated RRMS patients. Seroconversion was defined as at least two consecutive seropositive serum samples (or cessation of therapy after a single seropositive sample because of seropositivity) after initial seronegative testing. METHODS AND RESULTS: In our study of 179 patients for whom longitudinal blood samples were available over a long period (median 4.2 years), anti-JCV antibody indices were measured in 933 available samples. Eighty-six patients (48.0%) tested seronegative initially. Of these 86 seronegative patients, 23 patients (26.7%) seroconverted during follow-up. The annualized seroconversion rate was 7.1%. Seroconversion occurred between 9 and 90 months (median 43 months) of treatment. The rate of seroconversion was independent of follow-up duration. No significant increase was seen in the anti-JCV antibody index in the non-converting patients during the follow-up. CONCLUSION: The annualized seroconversion rate of 7.1% in patients using natalizumab, cumulatively leading to more than 25% of seronegative patients becoming seropositive in 4 years, is of clinical relevance and should be taken into account in the risk assessment when considering the start of natalizumab therapy.

Persistent spiking fever in a child with acute myeloid leukemia and disseminated infection with enterovirus.

We here report a 7 year old acute myeloid leukemia patient with persistent spiking fever likely caused by chronic echovirus 20 infection. After immunoglobulin substitution fevers subsided and the virus was cleared. Enterovirus infection should be considered in immunocompromised patients with unexplained persistent fever.

Urine is superior to saliva when screening for postnatal CMV infections in preterm infants.

BACKGROUND: Cytomegalovirus (CMV) is the most frequently contracted virus in preterm infants. Postnatal infection is mostly asymptomatic but is sometimes associated with severe disease. To diagnose an infection, urine or saliva samples can be tested for CMV-DNA by real-time polymerase chain reaction (rtPCR). Although the diagnostic accuracy of testing saliva samples has not been determined in preterm infants, saliva is widely used because it is easier to obtain than urine. OBJECTIVES: To determine whether screening of saliva is equivalent to urine to detect a postnatal CMV infection in preterm infants. STUDY DESIGN: Between 2010 and 2013 saliva and urine samples were collected from infants admitted to the Neonatal Intensive Care Unit of the University Medical Center Utrecht and born with a gestational age (GA) below 32 weeks. Urine samples were obtained within three weeks after birth and urine and saliva samples at term equivalent age (40 weeks GA) and tested for CMV-DNA by rtPCR. Infants with a congenital CMV infection were excluded. RESULTS: Of 261 preterm infants included in the study, CMV-DNA was detected in urine of 47 and in saliva of 43 children. Of 47 infants with postnatal CMV infection, CMV was detected in 42 saliva samples (sensitivity 89.4%; CI 76.9-96.5). Of 214 children without postnatal CMV infection, one saliva sample tested positive for CMV (specificity 99.5%; CI 97.4-99.9). CONCLUSIONS: Screening saliva for CMV-DNA by rtPCR is inferior to urine to diagnose postnatal CMV infections in preterm infants.

Leflunomide as part of the treatment for multidrug-resistant cytomegalovirus disease after lung transplantation: case report and review of the literature.

Treatment of cytomegalovirus (CMV) disease in transplant patients is challenging and, with antiviral resistance to first-line drugs, it remains uncertain which treatment algorithm to follow. Some data suggest that leflunomide, a pyrimidine synthesis inhibitor, can be used to treat resistant CMV infections. We report a 57-year-old CMV immunoglobulin-G (IgG)-seronegative woman, who received a bilateral lung transplant (LuTx) from a CMV IgG-positive donor with CMV primary disease. The CMV strain was genotypically resistant to ganciclovir, foscarnet, and cidofovir. After starting leflunomide as add-on therapy to a multidrug anti-CMV regimen, viral load declined substantially in 2 months without adverse events. This experience is discussed against the background of existing literature on the use of leflunomide as an anti-CMV agent in LuTx recipients.

A rare pulmonary infection caused by Arthrographis kalrae.

Arthrographis kalrae is a rare isolate in clinical specimens. Only ten cases of infection with this species have been described so far. To our knowledge, we report the first case of a pulmonary infection caused by A. kalrae in a patient with a past history of stage IIA Hodgkin's lymphoma and demonstrate that this organism can act as an opportunistic human pathogen.

Five-minute Giemsa stain for rapid detection of malaria parasites in blood smears.

The Giemsa stain is used as the gold standard for the diagnosis of malaria on blood smears. The classical staining procedure requires between 30 and 45 min. We modified the Giemsa stain and reduced the staining time to 5 min without any loss of quality.

Endosomal compartmentalization in three dimensions: implications for membrane fusion.

Endosomes are major sorting stations in the endocytic route that send proteins and lipids to multiple destinations in the cell, including the cell surface, Golgi complex, and lysosomes. They have an intricate architecture of internal membrane structures enclosed by an outer membrane. Recycling proteins remain on the outer membrane, whereas proteins that are destined for degradation in the lysosome are sorted to the interior. Recently, a retrograde pathway was discovered whereby molecules, like MHC class II of the immune system, return from the internal structures to the outer membrane, allowing their further transport to the cell surface for T cell activation. Whether this return involves back fusion of free vesicles with the outer membrane, or occurs via the continuity of the two membrane domains, is an unanswered question. By electron tomography of cryo-immobilized cells we now demonstrate that, in multivesicular endosomes of B-lymphocytes and dendritic cells, the inner membranes are free vesicles. Hence, protein transport from inner to outer membranes cannot occur laterally in the plane of the membrane, but requires fusion between the two membrane domains. This implies the existence of an intracellular machinery that mediates fusion between the exoplasmic leaflets of the membranes involved, which is opposite to regular intracellular fusion between cytoplasmic leaflets. In addition, our 3D reconstructions reveal the presence of clathrin-coated areas at the cytoplasmic face of the outer membrane, known to participate in protein sorting to the endosomal interior. Interestingly, profiles reminiscent of inward budding vesicles were often in close proximity to the coats.

Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography.

Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze-substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high-pressure frozen and aldehyde-fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.

Automated high-throughput electron tomography by pre-calibration of image shifts.

Electron tomography is a versatile method for obtaining three-dimensional (3D) images with transmission electron microscopy. The technique is suitable to investigate cell organelles and tissue sections (100-500 nm thick) with 4-20 nm resolution. 3D reconstructions are obtained by processing a series of images acquired with the samples tilted over different angles. While tilting the sample, image shifts and defocus changes of several microm can occur. The current generation of automated acquisition software detects and corrects for these changes with a procedure that incorporates switching the electron optical magnification. We developed a novel method for data collection based on the measurement of shifts prior to data acquisition, which results in a five-fold increase in speed, enabling the acquisition of 151 images in less than 20 min. The method will enhance the quality of a tilt series by minimizing the amount of required focus-change compensation by aligning the optical axis to the tilt axis of the specimen stage. The alignment is achieved by invoking an amount of image shift as deduced from the mathematical model describing the effect of specimen tilt. As examples for application in biological and materials sciences 3D reconstructions of a mitochondrion and a zeolite crystal are presented.

Interleukin-1 receptor type I gene-deficient mice are less susceptible to Staphylococcus epidermidis biomaterial-associated infection than are wild-type mice.

Elevated concentrations of interleukin-1 (IL-1) were found in tissue surrounding biomaterials infected with Staphylococcus epidermidis. To determine the role of IL-1 in biomaterial-associated infection (BAI), IL-1 receptor type I-deficient (IL-1R(-/-)) and wild-type mice received subcutaneous implants of silicon elastomer (SE) or polyvinylpyrrolidone-grafted SE (SEpvp), combined with an injection of 10(6) CFU of S. epidermidis or sterile saline. Neither mouse strain was susceptible to BAI around SE. IL-1R(-/-) mice with SEpvp implants had a no abscess formation and a reduced susceptibility to persistent S. epidermidis infection. The normal foreign body response, characterized by giant-cell formation and encapsulation, was delayed around SEpvp in wild-type mice but not in IL-1R(-/-) mice. This coincided with enhanced local IL-4 production in IL-1R(-/-) mice. These data suggest that inhibition of local IL-1 activity may be beneficial for the outcome of BAI.

Biomaterial-associated persistence of Staphylococcus epidermidis in pericatheter macrophages.

Biomaterial surfaces may be modified to reduce bacterial adhesion. The susceptibility in mice to Staphylococcus epidermidis infection in tissue surrounding the commonly used catheter materials-silicon elastomer (SE), polyamide (PA), and their surface-modified polyvinylpyrrolidone (PVP)-grafted derivatives, SE-PVP and PA-PVP, respectively-was assessed. Abscesses developed around SE-PVP. Around SE, PA, and PA-PVP catheters, no signs of infection were observed, although mice carrying PA-PVP developed septicemia after 14-21 days. S. epidermidis was cultured from the tissue surrounding PA-PVP segments. Cells around PA-PVP segments containing large numbers of bacteria were identified as macrophages by use of immunohistochemistry and electron microscopy. This persistence of intracellular bacteria was also observed around SE-PVP, SE, and PA catheters, although to a lesser extent. The cytokine profiles around the 4 materials were different. Implanted biomaterial induces an inflammatory response favorable to the persistence of S. epidermidis. Intracellular persistence of bacteria inside macrophages may be a pivotal process in the pathogenesis of biomaterial-associated infection.

Enhanced susceptibility to subcutaneous abscess formation and persistent infection around catheters is associated with sustained interleukin-1beta levels.

A persistent Staphylococcus epidermidis infection in mice around a subcutaneous polyvinylpyrrolidone-grafted silicon elastomer catheter (SEpvp) but not around a conventional silicon elastomer catheter was observed. With SEpvp pericatheter tissue, protracted and exaggerated interleukin-1beta (IL-1beta) production was found. Apparently, sustained levels of IL-1beta are associated with enhanced susceptibility to biomaterial-associated S. epidermidis infection.